Graphene Oxide Nanosheets Toxicity in Mice Is Dependent on Protein Corona Composition and Host Immunity

Two-dimension graphene oxide (GO) nanosheets with high and low serum protein binding profiles (high/low hard-bound protein corona/HChigh/low) are used in this study as model materials and screening tools to investigate the underlying roles of the protein corona on nanomaterial toxicities in vivo. We proposed that the in vivo biocompatibility/nanotoxicity of GO is protein corona-dependent and host immunity-dependent. The hypothesis was tested by injecting HChigh/low GO nanosheets in immunocompetent ICR/CD1 and immunodeficient NOD-scid II2rγnull mice and performed histopathological and hematological evaluation studies on days 1 and 14 post-injection. HClow GO induced more severe acute lung injury compared to HChigh GO in both immunocompetent and immunodeficient mice, with the effect being particularly pronounced in immunocompetent animals. Additionally, HClow GO caused more significant liver injury in both types of mice, with immunodeficient mice being more susceptible to its hepatotoxic effects. Moreover, administration of HClow GO resulted in increased hematological toxicity and elevated levels of serum pro-inflammatory cytokines in immunocompromised and immunocompetent mice, respectively. Correlation studies were conducted to explore the impact of distinct protein corona compositions on resulting toxicities in both immunocompetent and immunodeficient mice. This facilitated the identification of consistent patterns, aligning with those observed in vitro, thus indicating a robust in vitro–in vivo correlation. This research will advance our comprehension of how hard corona proteins interact with immune cells, leading to toxicity, and will facilitate the development of improved immune-modulating nanomaterials for therapeutic purposes.

DCFDA dilution per well, then continually incubated for the 45min.Fluorescence intensity was detected by fluorescence spectroscopy with maximum excitation and emission spectra of 485nm and 535nm, respectively, in a FLUOstar Omega microplate reader (BMG Labtech,   Germany).Folds increased in ROS production was calculated using the following equation: (Ftest-Fblank2)/(Fcontrol-Fblank1).
Cells were maintained in a T75 cell culture flask until 80% confluency (37˚C, 5% CO2) before seeding into 6-well plates.J774 cells were seeded in different 6-well plates (3 x 10 5 /well) 24 h before the incubation with graphene samples.Freshly prepared graphene samples (HCHigh GO and HCLow GO) at 10, 50, and 100 µg/mL were used and incubated with the cells for 24 h (1 mL/well).H2O2 was used as a positive control at a concentration of 2.43 mmol/L.After 24 h incubation, ice-chilled PBS gently rinsed the remaining still viable cells.Nonadherent dead cells were aspirated with PBS (1 mL/well).Rinsed viable cells were trypsinised by 300 µL 0.05% trypsin-EDTA at 37˚C for 3 min before adding the serum-containing cell culture media to stop the trypsinisation (500 µL/well).Cell suspensions were transferred into 2 mL centrifuge tubes and centrifuged at 4,000 rpm (Beckman Coulter Allegra X-22R Benchtop Centrifuge, rotor: F2402H) for 10 min at 4˚C.The supernatant was removed.
The washed cell pellets were then resuspended in 100 uL 5% 5-Sulfosalicylic Acid (SSA) and frozen-thawed twice to obtain the cell lyse.The cell lysates were centrifuged at 14,000 rpm (Eppendorf 5810R, rotor: F45-30-11) for 10 min at 4˚C to obtain the supernatant for GSH activity evaluation using Glutathione Assay Kit in 96-well plates.
In brief, the two reagent blank samples comprised 5% SSA (10 µL) and a working mixture (150 µL).Glutathione Standard Solutions were prepared: various dilutions of 10 µL samples of the prepared Glutathione Standard Solutions+150 µL working mixture.The unknown samples were constituted of the unknown sample in duplicate (X µL), 5% SSA (up to 10 µL sample), and a working Mixture (150 µL).After Incubating for 5 minutes at room temperature, these three samples were added 50µL NADPH (0.16 mg/mL) and then mixed.The UV absorbance of each well was measured at 412 nm using a microplate reader (FLUOstar Omega, BMG Labtech) with Omega software (v2.1).The standard curve was determined by the values of the Glutathione Standard Solutions, then the △A412/min equivalent to 1 nmole of reduced glutathione per well was calculated.The nmoles of GSH in the unknown sample were calculated using the following equation: The nmoles of GSH in the unknown sample=(△A412/minn(sample)×dil)/ (△A412/minn(nmoles) ×vol) where dil represents the dilution factor of the original sample and vol represents the volume of sample in the reaction in mL.The % GSH depletion was calculated by normalizing to the untreated control cells.
J774 cells were seeded in different 12-well plates (8 x 10 4 /well) 24 h before the incubation with graphene samples.Freshly prepared graphene samples HCHigh GO and HCLow GO) at 10, 50, and 100 µg/mL were used and incubated with the cells for 24 h (1 mL/well).H2O2 was used as a positive control at a concentration of 2.43 mmol/L.After 24 h incubation, ice-chilled PBS gently rinsed the remaining still viable cells.Nonadherent dead cells were aspirated with PBS (1 mL/well).Rinsed viable cells were trypsinised by 300 µL 0.05% trypsin-EDTA at 37˚C for 3 min before adding the serum-containing cell culture media to stop the trypsinisation (500 µL/well).Cell suspensions were transferred into 2 mL centrifuge tubes and centrifuged at 4,000 rpm (Beckman Coulter Allegra X-22R Benchtop Centrifuge, rotor: F2402H) for 10 min at 4˚C.The supernatant was discarded.The washing step was repeated twice (1.5 mL PBS/tube).The washed cell pellets were resuspended in 200 μL PBS and frozen-thawed twice to obtain the cell lyse.The cell lysates were centrifuged at 14,000 rpm (Eppendorf 5810R, rotor: F45-30-11) for 15 min at 4˚C to obtain the supernatant for SOD activity evaluation using the SOD determination kit in 96-well plates.
In brief, three types of blank control samples were prepared: Blank 1 (20 µL double distilled The samples were constituted of cell lyse (20 µL), WST working solution (200 µL), and enzyme working solution (20 µL).The samples and the blank controls were mixed thoroughly within the 96-well plate and then incubated at 37 °C for 20 min.The UV absorbance of each well was measured at 440 nm using a microplate reader (FLUOstar Omega, BMG Labtech) with Omega software (v2.1).The SOD activity was calculated using the following equation: × 100%.The relative SOD activity was calculated by normalizing the untreated control cells.

Assessment of cellular viability by a modified lactate dehydrogenase (mLDH) assay
The modified lactate dehydrogenase (mLDH) assay was used, i.e., instead of measuring the LDH released, the LDH remained within the cell and was analysed to minimise the interference from the graphene [10,16] .J774 cells (1 x 10 4 /well) were seeded onto 96-well plates and allowed to be set for 24 h.Freshly prepared graphene samples (HCHigh GO and HCLow GO) at 10, 50, and 100 µg/mL were used and incubated with the cells for 24 h.DMSO (10% in cell culture media) was used as a positive control for the assay, as it causes cytotoxicity by interfering with membrane permeability.After 24 h incubation, cell culture media was carefully and slowly removed using multichannel pipettes.The remaining cells were lysed in phenol-red and serum-free Adv.RPMI containing 0.9% Triton X-100 at 37˚C for 1h (100 µL/well).The cell lysates were centrifuged at 4000 rpm (Eppendorf 5810 R) at 4˚C for 1 h to precipitate the graphene within the cell lyse.The supernatant was carefully recovered (to avoid graphene samples precipitate), and the CytoTox96 ® LDH assay kit was used for cytotoxicity assay, following the manufacturer's instructions.Absorbance at 490 nm was measured in the FLUO star Omega microplate reader (BMG Labtech, Germany).Cell viability was calculated as the percentage of control untreated cells using the following equation: {(A490 of treated cells -A490 of negative control)/(A490 of untreated cells-A490 of negative control)} × 100.Negative control: phenol-free Adv.RPMI containing 0.9% Triton X-100.

Supporting Results
The pro-oxidative potential of HCHigh/Low GO HCLow GO, with strongly pro-oxidative potential, produced more ROS than HCHigh GO.

Figure S1 .
Figure S1.Assessing the pro-oxidative potential and cytotoxicity of HCHigh/Low GO.Prooxidative potential assessment of HCHigh/Low GO in J774 cells by ROS, GSH, and SOD assay.Cytotoxicity of HCHigh/Low GO was assessed by the modified lactate dehydrogenase (mLDH) assay.After exposure to HCHigh/Low GO for 24h, more ROS was produced, and GSH depletion was observed.Lower relative SOD activities and cell viability were observed only in HCLow GO-treated J774 cells, while no other changes were observed in HCHight GO-treated J774 cells.Data expressed as mean values ± standard deviation (SD), n = 4. * p<0.05,** p<0.01, *** p<0.001 compared to the untreated group.

Figure S2 .
Figure S2.Organ Coefficients of Heart, Spleen, and Kidney in ICR/CD1 and NOD-scid Il2ry null mice on Days 1 and 14 after IV Injections of HCHigh/Low GO.

Figure S7 .
Figure S7.Clinical Biochemistry Results from NOD-scid II2rγ null Mice Treated with HCHigh/Low GO.Healthy NOD-scid II2rγ null mice were single-dose treated intravenously with HChigh/low GO at the dose of MTD.Half of the animals in each group were assessed on the day following dosing, and the remaining animals were assessed on day 14.Serum biochemistry profiles of NOD-scid II2rγ null mice exposed to HCHigh/Low GO were analysed at 1-and 14-day post-exposure.A decrease of AST/ALT and higher ALP were observed in HCLow GO-treated mice at 1-day post-exposure.Data are expressed as mean values ± standard deviation (SD), n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the untreated group; # p <0.05 compared to HCHigh GO.

Table S4 . Correlation Analysis Between Protein Corona and Relative Lung Infiltration (%)
value <0.05, it was considered there were an association between protein corona and lung infiltration.rS-values close to -1 or +1 represent stronger relationships than values closer to zero.